Project Summary The overall goal of this proposal is to facilitate my aspiration to become a primary investigator of chromosome biology at a research-intensive university. This project will build on my graduate background in mammalian meiosis while expanding my technical abilities to facilitate my investigation of chromosome biology in other contexts, such as human disease. The proposed research utilizes S. cerevisiae to investigate the chromosome dynamics that occur prior to the first meiotic division. During this time, the nucleus is reorganized to allow for the alignment of homologous chromosomes. The proper alignment of homologous chromosomes depends on a series of molecular pathways, including endogenous DNA damage and repair, checkpoint signaling, and the construction of a structural scaffold known as the synaptonemal complex (SC). The SC is made of two lateral elements that form along the lengths of each chromosome and one central element that connect the lateral elements of homologous chromosomes. The conserved axis proteins Hop1 (HORMAD1/2 proteins in mice and humans) are an essential component of the lateral element and a central regulator of SC assembly. Despite extensive research, how the axis proteins localize to chromatin and then recruit the central element to build the SC remains unclear. Indeed, a large fraction of Hop1 dissociate from the lateral elements as the central element is deposited, raising the question whether Hop1 is a structural component of the SC or only needed to mediate deposition of the central element. Aim 1 utilizes conditional protein induction and depletion experiments to test the requirements of Hop1 in SC formation. It will also determine the role of Hop1 dissociation in this process. Aim 2 investigates the interface between DNA and the lateral element by using a novel hop1 separation-of-function allele that exhibits normal DSB induction and repair kinetics but altered DNA breakage patterns, which likely reflect altered Hop1 chromatin association. This allele of HOP1 removes an uncharacterized region in the center of Hop1 that shares features with PHD domains, which are canonically important for reading and responding to histone marks. Aim 2-1 uses this hop1 mutant to determine the effects the axis patterning by ChIP-seq of axis proteins and to correlate these effects with altered break patterning observed by whole-chromosome Southern blot analysis. Aim 2-2 utilizes targeted biotinylation and mass spectrometry to define the chromatin environment surrounding Hop1 binding sites. This experiment will be used to identify candidate proteins that will be tested for a role in axis protein positioning using the yeast deletion library. The results of this proposal will provide important new insight into the mechanism of meiotic chromosome morphogenesis and will serve as a framework for understanding infertility and birth defects in humans. This proposal has a strong training component and will expand my knowledge to whole genome sequencing techniques, computational biology, and proteomics. Altogether, this training will provide me with the experience needed for establishing an independent research program in chromosome biology.